CN116536409B - Three-dimensional genome detection method compatible with wide-range ChIP DNA input amount - Google Patents
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Abstract
The invention provides a three-dimensional genome detection method compatible with the input amount of wide-range ChIP DNA, which can break ChIP DNA with different input amounts within a certain range through single enzyme digestion reaction of fragmenting enzyme, connect a sequencing joint at the tail end of the broken ChIP DNA, capture three-dimensional interaction ChIP DNA fragments through biotin enrichment, amplify and screen to obtain a good library, and obtain a three-dimensional genome interaction mode through sequencing analysis such as a high-throughput second-generation sequencer. Because the ChIP DNA yield of a single sample is generally within the input range of compatible fragmenting enzymes, the method can finish the breaking treatment of all ChIP DNAs of the single sample only by a single enzyme digestion reaction, reduces the times of enzyme digestion reaction and experimental cost, and has excellent data quality.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a three-dimensional genome detection method based on ChIP DNA.
Background
Eukaryotic chromatin assumes a highly folded state in the nucleus, and its folded spatial structure is closely related to gene expression. Thus, three-dimensional genomics, which is based on the spatial structure of chromatin and its functions, is becoming a new break through for understanding the vital activities, ontogeny and disease occurrence mechanisms of cells.
Chromatin interaction analysis techniques (chromatin interaction analysis using paired-end tag sequencing, chIA-PET) based on paired-end tag sequencing integrate chromatin co-immunoprecipitation (Chromatin immunoprecipitation, chIP) and chromatin conformation capture techniques, and can capture ChIP DNA where chromatin interactions occur, thereby identifying specific protein-mediated chromatin interactions across the whole genome, and are classical detection methods for three-dimensional genomics research. In recent years in situ ChIA-PET has greatly improved the capture efficiency of three-dimensional genome interaction modes by performing chromatin cleavage and ligation reactions in situ in the nucleus, but the experimental difficulty and cost remain high, which is mainly reflected in the later library processing part.
At present, in situ ChIA-PET adopts transposase to carry out enzyme digestion and disruption construction of the captured ChIP DNA in the post-treatment process. At present, a single transposase reaction can only finish the breaking treatment of DNA with a specific input amount (such as 50 ng). However, the use of different cell types, numbers, and antibodies recognizing different antigens can affect the yield of ChIP DNA, ranging from a few nanograms to hundreds of nanograms. In a single transposase reaction, insufficient or too high an input of DNA often results in library construction failure. Many times of transposase cleavage reactions are often required to break a portion of the ChIP DNA in the late stages of current in situ ChIA-PET experiments, while a portion of ChIP DNA may remain insufficient to react the minimum amount of input required. The post-processing method can not fully utilize all ChIP DNA of a single sample, and increases the experiment times, the operation difficulty and the reagent consumable cost. In order to promote ChIA-PET applicability, it is necessary to develop an easy-to-operate, low-cost post-processing method compatible with a wide range of ChIP DNA inputs.
Disclosure of Invention
The inventor researches and discovers that for all ChIP DNA of which samples are obtained through pretreatment, chIP DNA can be broken at one time through a single enzyme digestion reaction, then a sequencing joint is connected to the tail end of the DNA, a three-dimensional interaction DNA region is captured through biotin enrichment, amplification and screening are carried out, a good library can be obtained, and a three-dimensional genome interaction mode can be obtained through sequencing analysis such as a high-throughput second-generation sequencer, so that the invention is completed. The invention is compatible with the wide-range ChIP DNA input amount, can finish the breaking treatment of all ChIP DNAs of a single sample only by one enzyme digestion reaction, reduces the times of enzyme digestion reaction and experimental cost, and has excellent data quality.
The invention aims to provide a three-dimensional genome detection method compatible with a wide-range ChIP DNA input amount, which adopts a fragmenting enzyme to carry out a one-time enzyme digestion reaction on ChIP DNA obtained by in situ ChIA-PET pretreatment so as to interrupt all ChIP DNA of a single sample at one time.
In the present invention, the term "in situ ChIA-PET" refers to one way of chromatin interaction analysis technique (chromatin interaction analysis using paired-end tag sequencing) based on paired-end tag sequencing, i.e., in situ chromatin interaction analysis based on paired-end tag sequencing.
The term "ChIP DNA" obtained by in situ ChIA-PET pretreatment refers to ChIP DNA obtained by conventional in situ ChIA-PET pretreatment such as cell crosslinking, cell lysis, restriction, chromatin tailing and ligation, and chromatin co-immunoprecipitation (shown in fig. 1).
In the invention, the reaction of the fragmenting enzyme and the total ChIP DNA generated by a single sample is disposable, and multiple reactions are not needed to complete the fragmenting process of all ChIP DNA, thereby greatly simplifying the operation.
In the present invention, preference is given to the fragmenting enzymes FEA Enzyme Mix (Norweizan), smearase (next holy), KAPA flag Enzyme (KAPA), NEBNEext DSDNA FRAGMENTASE (NEB), NEBNext Ultra II FS Enzyme Mix (NEB), and other fragmenting enzymes containing non-specific nuclease components, more preference being given to the fragmenting enzymes FEA Enzyme Mix (Norweizan), smearase (next holy), KAPA flag Enzyme (KAPA), NEBNEext DSDNA FRAGMENTASE (NEB), or NEBNext Ultra II FS Enzyme Mix (NEB).
In a preferred embodiment of the present invention, the amount of the enzyme to be added is 2 to 10. Mu.L, and the amount of ChIP DNA to be added obtained by the in situ ChIA-PET pretreatment is in the range of 0.1 to 1000ng. In a more preferred embodiment of the present invention, the amount of the enzyme to be added is 10. Mu.L, and the amount of ChIP DNA obtained by the in situ ChIA-PET pretreatment is in the range of 1 to 100ng.
In a preferred embodiment of the present invention, the process of performing the cleavage reaction with the fragmenting enzyme comprises: reacting for 2-30 min at 25-37 ℃, then reacting for 10-30 min at 65-72 ℃, and finally reacting for 5-10 min at 4-10 ℃. In a more preferred embodiment of the present invention, the process of the cleavage reaction of the mixture of fragmenting enzymes comprises: the reaction is carried out for 2 to 6 minutes at 37 ℃, then for 10 minutes at 65 ℃ and finally for 5 minutes at 4 ℃.
The ChIP DNA obtained by in situ ChIA-PET pretreatment is interrupted once by the appropriate/compatible reaction temperature and reaction time of the above-described fragmenting enzyme. After cleavage of the ChIP DNA obtained by in situ ChIA-PET pretreatment by a fragmenting enzyme into small fragments, a sequencing adapter was ligated to the DNA end. The method is not particularly limited, and a sequencing linker commonly used in the art may be used to join the sequencing linker by a conventional bridging reaction.
In the present invention, avidin-coupled magnetic beads are subjected to a biotin enrichment reaction to capture ChIP DNA containing biotin labels that undergo three-dimensional interactions.
In the present invention, the term "three-dimensional interaction" has the same meaning as "three-dimensional interaction".
Library amplification was performed on target DNA fragments captured by biotin enrichment. The amplification method is not particularly limited, and a usual amplification method, means or method can be used.
In a preferred embodiment of the invention, the DNA fragments after amplification of the library are distributed over a length of between 200 and 1000 bp.
It is further preferred that the library amplification products are fragment screened using BluePippin and/or DNA extraction magnetic beads, the fragment ranges from 280 to 580bp, and other fragment ranges compatible with second generation sequencing, so that the library amplification products screened for fragments are sequenced using a second generation sequencer.
Another object of the present invention is to provide a in situ ChIA-PET late library processing method suitable for a wide range of ChIP DNA input amounts, which uses a fragmenting enzyme to perform one-time disruption on ChIP DNA obtained by in situ ChIA-PET early processing to generate small fragment DNA.
In the application, the three-dimensional genome detection method compatible with the wide-range ChIP DNA input amount or the in situ ChIA-PET later library processing method applicable to the wide-range ChIP DNA input amount is referred to by simply called as 'fragment-ChIA-PET'.
The method of the invention has the following characteristics/advantages:
(1) The method comprises the steps of adopting fragmenting enzyme, completing breaking treatment of all ChIP DNA of in situ ChIA-PET single samples through single reaction, and then carrying out biotin enrichment, sequencing joint addition, library amplification, fragment screening and sequencing analysis to detect three-dimensional genome structure information mediated by protein;
(2) Compared with in situ ChIA-PET post-treatment which requires multiple transposase reactions, the method only needs single enzyme digestion breaking reaction of the fragmented enzyme, and can meet the library construction requirement of ChIP DNA with wide input quantity. The enzyme and the related reagent consumable materials with various specifications are not required to be selected and matched according to different ChIP DNA input amounts, and only the fragmented enzyme with single specification and the related reagent consumable materials are required, so that the waste of experimental consumable materials is avoided, and the experimental operation difficulty and the experimental cost of ChIA-PET post-treatment are reduced.
(3) Compared with in situ ChIA-PET post-treatment which can leave a part of ChIP DNA which does not meet the minimum input amount required by the transposase reaction, the invention can fully utilize all ChIP DNA produced by a single sample, especially for a small amount of precious samples;
(4) The invention reduces the difficulty and cost of experimental operation, and simultaneously still maintains the data quality basically consistent with in situ ChIA-PET, and even better performs on individual indexes.
Drawings
FIG. 1 shows a flow chart of the method of the present invention;
FIG. 2 shows the distribution of DNA length during the library amplification and fragmentation screening stage in example 1 of the present invention;
FIG. 3 shows statistics of Frag-ChIA-PET sequencing data in example 1 of the present invention;
FIG. 4 shows the visualization of the Frag-ChIA-PET sequencing data in example 1 of the present invention.
Detailed Description
The features and advantages of the present invention will become more apparent and evident from the following detailed description of the invention.
Example 1
1.1 Overview:
(1) To illustrate the effect of different ChIP DNA inputs on library construction over a wide range, in human leukemia cell line K562, the fragment-ChIA-PET library of the present invention was constructed using 1ng, 10ng, and 100ng of RNA polymerase II (RNAPII) targeted ChIP DNA, respectively, and compared.
(2) To demonstrate the data quality of the Frag-ChIA-PET of the present invention, a in situ ChIA-PET library was constructed in K562 cells using 150ng of RNAPII targeted ChIP DNA and compared to the library data described above.
(3) To demonstrate the portability of the Frag-ChIA-PET of the invention to different antigen applications, libraries of the invention were constructed in K562 cells using 5.5ng and 7.8ng of histone H3K4me 3-targeted ChIP DNA, respectively, and library quality comparisons were made.
1.2 Test procedure
1.2.1 Pretreatment
(1) Cell cross-linking:
Human leukemia cell line K562 was cross-linked with 1% formaldehyde at room temperature for 20 min, neutralized with 0.2M glycine and washed with 1 XDPBS, followed by a second cross-linking with 1.5mM Ethylene glycol-bis (succinic acid N-hydroxysuccinimide ester) at room temperature for 45 min to fix the chromatin in the nuclei sufficiently.
(2) Cell lysis:
Taking a 2-4×10 6 crosslinked K562 cell sample, and firstly reacting for 1 hour at 4 ℃ by using 100 mu L of 0.1% SDS; then, 100. Mu.L of 0.55% SDS was used instead, and the reaction was carried out at room temperature for 10 minutes, at 62℃for 10 minutes, and at 37℃for 10 minutes, respectively. Finally, 270. Mu.L of water without ribozyme and 50. Mu.L of 10% Triton X-100 were used to neutralize the cell lysate.
(3) Restriction enzyme cutting:
50. Mu.L of 10X CutSmart buffer (NEB) was added to the cell-neutralized solution, and after mixing, 30. Mu.L of 10U/. Mu.L of restriction enzyme Alu I (NEB) was added thereto, and the reaction was carried out overnight at 37℃to thereby cleave the chromatin sufficiently in situ at the nucleus and expose the blunt ends of the chromatin.
(4) Chromatin tailing and ligation
To the cells after the cleavage, 4. Mu.L of 10x CutSmart buffer (NEB), 12. Mu.L of 20mg/mL BSA (Takara), 12. Mu.L of 10mM dATP (NEB), and 12. Mu.L of 5U/. Mu.L Klenow (3 '. Fwdarw.5' exo-) (NEB) were added, and reacted at room temperature for 1 hour to add A tail at the blunt end of chromatin. Subsequently, 200. Mu.L of 5X quick ligase buffer (NEB), 260. Mu.L of water without ribozyme, 3. Mu.L of 200 ng/. Mu.L of biotin-labeled bridge linker, and 11. Mu.L of 400U/. Mu.L of T4 DNA LIGASE (NEB) were added, and the reaction was carried out overnight at 16℃to effect ligation of chromatin fragments in situ in the nucleus.
(5) Chromatin co-immunoprecipitation (ChIP)
Crushing the connected chromatin by adopting an ultrasonic crusher, incubating the magnetic beads coupled with protein A and/or protein G with RNAPII or histone H3K4me3 antibody, and performing chromatin co-immunoprecipitation reaction on the magnetic beads combined with the antibody and the crushed chromatin to obtain the RNAPII or H3K4me3 targeted ChIP DNA.
1.2.2 Post-treatment
1.2.2-1 Post-treatment of conventional in situ ChIA-PET
Post-treatment of conventional in situ ChIA-PET was performed on RNAPII-targeted ChIP DNA at an input of 150 ng.
Specifically, in each transposase cleavage reaction, 50ng of purified ChIP DNA was taken, added with water without ribozyme to make up to 35 μl, then 10 μl of buffer 5×ttbl (novenan) and 5 μl of transposase TTE Mix V50 (novenan) were added, respectively, and after mixing, the cleavage reaction was performed. The reaction time was 55℃for 10 minutes.
To fully perform the ChIP DNA disruption reaction, 3 transposase (nuezan) cleavage reactions with 50ng DNA input size were required, 3 reagent consumables were consumed, and then subsequent DNA library construction, sequencing and analysis procedures were performed.
If the single sample ChIP DNA input is not enough to require a minimum 50ng input for the Tn5 transposase reaction, normal pooling will not be performed, resulting in wastage of the original ChIP DNA material.
1.2.2-2 Post-treatment of Frag-ChIA-PET according to the invention
(1) Disposable fragmenting enzyme reaction
The ChIP DNA obtained by the pretreatment is extracted, and then the ChIP DNA with different input amounts and different antibody sources is subjected to one-time breaking reaction of the fragmenting enzyme.
Specifically, all ChIP DNA of the single sample after purification was taken, and water without ribozyme was added to make up to 35 μl, and then 5 μl Buffer FEA Buffer (nuuzan) and 10 μl of the fragmenting Enzyme FEA Enzyme Mix (nuuzan) were added, respectively, and after mixing, the fragmenting reaction was performed. The reaction parameters were 37℃for 2 to 6 minutes, 65℃for 30 minutes, and 4℃for 5 minutes in this order.
As shown in FIG. 2, the 1ng, 10ng and 100ng RNAPII-targeted ChIP DNA and the 5.8ng and 7.7ng H3K4me 3-targeted ChIP DNA fragmenting enzyme reactions in this example were all one-time reactions.
(2) Joint sequencing connector
To the 50. Mu.L of the reaction product, the following reagents required for the ligation reaction were added in order: 15. Mu.L of non-ribozyme water, 25. Mu L Rapid ligation buffer (Nor Renz), 5. Mu. L DNA ADAPTER (Nor Renz), and 5. Mu. L RAPID DNA LIGASE (Nor Renz). After mixing, the reaction was carried out with parameters of 20℃for 15 minutes and 4℃for 5 minutes in this order.
(3) Biotin enrichment
The fragmented and sequencing linker added ChIP DNA was extracted using Zymo Genomic DNA Clean & Concentrators kit.
The resulting DNA product contains biotin-labeled three-dimensional interacted DNA, and biotin-free non-three-dimensional interacted DNA. Thus, there is a need to further enrich for biotin-labeled target DNA using avidin-conjugated magnetic beads.
Next, 30. Mu.L of streptavidin-coupled magnetic beads were pre-treated for each sample. The beads were first washed with buffer containing 2M NaCl, 0.5mM EDTA and 5mM Tris-HCl and treated with I-block for 1 hour at room temperature to block non-specific macromolecular binding sites. Then washed with a buffer containing 1M NaCl, 0.5mM EDTA and 5mM Tris-HCl, and treated with biotin-free genomic DNA fragments for 1 hour at room temperature to block potential non-specific nucleic acid binding sites. The purified DNA product was then diluted 1:1 by volume with a buffer of 2M NaCl, 0.5mM EDTA and 5mM Tris-HCl, and the dilution was added to the pretreated beads and allowed to bind for 1 hour at room temperature. Then washed sequentially with a washing solution containing 2 XSSC and 0.5% SDS and a buffer containing 1M NaCl, 0.5mM EDTA and 5mM Tris-HCl, and finally the magnetic beads to which the biotin-labeled target DNA was bound were resuspended in 20. Mu.L of nuclease-free water or EB buffer.
(4) Library amplification
The above 20. Mu.L of the magnetic beads to which the target DNA labeled with biotin was attached were transferred to a 0.2mL of an EP tube without ribozyme, followed by sequentially adding 5. Mu. L PCR PRIMER Mix 3 (Nuo 'zan) and 25. Mu. L VAHTS HIFI Amplification Mix (Nuo' zan). After mixing, the following library amplification reactions were performed: a single reaction was carried out at 95℃for 3 minutes, a multiple cycle reaction was carried out at 98℃for 20 seconds, 60℃for 15 seconds and 72℃for 30 seconds, a single reaction was carried out at 72℃for 5 minutes, and a single reaction was carried out at 4℃for 5 minutes.
Wherein: 18, 17 and 19 cycles of cycling reactions were performed on 1ng, 10ng and 100ng RNAPII targeted ChIP DNA libraries, respectively; 17 cycles of reactions were performed on 5.8ng and 7.7ng of H3K4me3 targeted ChIP DNA library.
Library DNA was extracted using AMPure XP DNA extraction beads (Beckman). Finally, agilent 2100 was used to detect the fragment distribution of the amplified library.
As shown in FIG. 2a, the amplified lengths of the ChIP DNA library with different input amounts and different antigen targets are mostly concentrated in the range of 200-1000bp, and meet the requirement of second-generation sequencing.
(5) Fragment screening
To further obtain high quality sequencing data, bluePippin was used to fragment the amplified library.
2% Agrarose GEL CASSETTE prefabricated gel boxes and V1 internal reference markers are selected, and fragment screening parameters are 280-580 bp. The length of the screened DNA fragments was checked using Agilent 2100.
As shown in FIG. 2b, the final DNA fragments were distributed over a range of screening parameters and the like, meeting the requirements of on-machine sequencing.
(6) Sequencing analysis
A second generation sequencer such as Hiseq X Ten from Illumina company is used for sequencing with 150bp double ends, chIA-PIPE is used for data analysis, and Basic browser is used for visual display of results.
As shown in fig. 3:
① The fragment-ChIA-PET library containing bridge linker for RNAPII targeted 1ng, 10ng and 100ng ChIP DNA had a reads ratio (fraction_read_ pairs _with_linker) of greater than 90%, slightly higher than the in situ ChIA-PET library (88%) for RNAPII targeted 100ng ChIP DNA, indicating high capture rate of three-dimensional interactive DNA fragments for fragment-ChIA-PET.
② The ratio of the intra-chromatin interactions with paired tags (PET) or clusters (cluster) of Frag-ChIA-PET was greater than 1, consistent with the results for in situ ChIA-PET, indicating that the final available valid data was high.
In addition, to demonstrate the portability of the present invention, fragment-ChIA-PET library construction was performed on histone H3K4me3 targeted 5.5ng and 7.8ng ChIP DNA.
Statistical results show that H3K4me3 targeted fragment-ChIA-PET library also has a higher ratio of reads containing bridge linker and a ratio of interactions between chromatin interior and exterior. From the statistical results, the library quality of the ChIP DNA construction of the fragment-ChIA-PET over a wide input amount is consistent with that of in situ ChIA-PET, even better, and the example demonstrates that the method can be transplanted to the ChIA-PET library construction of ChIP DNA from different antigen sources.
As shown in FIG. 4, from the visual results, the RNAPII and H3K4me3 targeted fragment-ChIA-PET libraries can both detect obvious chromatin interaction patterns, and the interaction patterns in the same genomic region are more consistent, further demonstrating that the library production data has good visual quality. Wherein the high input ChIP DNA library has more abundant genome visualization details, further proving the necessity of making full use of all ChIP DNA of a single sample for subsequent library construction.
The invention has been described in detail in connection with the detailed description and the exemplary examples and the accompanying drawings, but the description is not to be construed as limiting the invention. It will be understood by those skilled in the art that various equivalent substitutions, modifications or improvements may be made to the technical solution of the present invention and its embodiments without departing from the spirit and scope of the present invention, and these fall within the scope of the present invention. The scope of the invention is defined by the appended claims.
Claims (7)
1. A three-dimensional genome detection method compatible with wide-range ChIP DNA input amount adopts a single enzyme digestion reaction of ChIP DNA obtained by in situ ChIA-PET pretreatment by adopting a fragmenting enzyme, and completes the breaking treatment of all ChIP DNA of a single sample at one time,
The fragmenting Enzyme is FEA Enzyme Mix,
The enzyme digestion reaction process of the fragmenting enzyme comprises the following steps: reacting for 2-30 min at 25-37 ℃, then reacting for 10-30 min at 65-72 ℃, finally reacting for 5-10min at 4-10 ℃,
The dosage of the fragmenting enzyme is 10 mu L, and the dosage of ChIP DNA obtained by in situ ChIA-PET pretreatment is 1-100 ng.
2. The method of claim 1, wherein said in situ ChIA-PET pretreatment results in ChIP DNA obtained by cell cross-linking, cell lysis, restriction, chromatin tailing, and chromatin co-immunoprecipitation.
3. The method of claim 1, further comprising the step of, after interrupting ChIP DNA:
(2) Ligating a sequencing adapter to the broken ChIP DNA end;
(3) Biotin enrichment reaction was performed using avidin-coupled magnetic beads to capture three-dimensional interactive DNA containing biotin labels.
4. A method as claimed in claim 3, further comprising the steps of:
(4) Amplifying a target DNA library;
(5) Selecting amplification products with proper length range from the library;
(6) Library sequence information was obtained by a high throughput second generation sequencer and three dimensional genomic interaction pattern was obtained by bioinformatic analysis.
5. The method of claim 4, wherein the length of the amplified DNA fragments of the library is mainly between 200 and 1000 bp.
6. The method of claim 4, wherein the library amplification products are subjected to fragment screening using BluePippin and/or DNA extraction beads, the fragment ranges from 280 to 580 bp, and other fragment ranges compatible with second generation sequencing.
7. A method of in situ ChIA-PET post library treatment suitable for a wide range of ChIP DNA inputs comprising the steps of any one of claims 1-6.
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